The long term goal of the proposed studies is to understand the intracellular signaling mechanisms in the regulation of cell cycle progression and other cellular functions in diseases such as cancer. The previous funding period focused on the role of focal adhesion kinase (FAK) and its downstream signaling pathways in mediating integrin regulation of cell cycle progression. We have identified a number of FAK downstream pathways for its regulation of cell cycle progression and also showed the critical importance of focal contacts localization of the FAK signaling complexes. We have found that upregulation of cyclin D1 by FAK is primarily responsible for FAK-stimulated cell cycle progression and that FAK regulates cyclin D1 expression through Erk signaling pathways and subsequent activation of Ets transcription factor binding to the EtsB site in the cyclin Dl promoter. Lastly, we have identified the transcription factor KLF8 as a mediator of FAK regulation of cyclin Dl and cell cycle progression. These studies also identified a novel protein inhibitor for FAK called FIP200 (pAK-family Interacting Protein of 200 kDa). FIP200 binds directly to the kinase domain of FAK and inhibits its kinase activity, which correlated with FIP200 inhibition of various cellular functions including cell cycle progression. We have identified an interaction between FIP200 and TSC complex, which may regulate TSC activity and its downstream pathways. We also found that FIP200 regulated expression of cyclin Dl and p21, both of which contributed to cell cycle regulation by FIP200. Lastly, to study potential functions of FIP200 in vivo, we prepared both heterozygous total FIP200 knockout (KO) mice and floxed FIP200 mice for tissue specific KO in preliminary studies. In this renewal, we plan to focus on the analysis of FIP200 in signal transduction and its potential tumor suppressor functions in vivo. Aim 1 will study the mechanism of FIP200 regulation of TSC complex and potential role of this interaction in the regulation of cell cycle progression, cell survival and cell size control. Aim 2 will determine the mechanism of FIP200 regulation of cyclin Dl and p21 in cell cycle regulation by FIP200. Aim 3 will investigate the in vivo functions of FIP200 using both FIP200 null mouse embryonic fibroblasts and mammary gland specific FIP200 conditional KO mice with emphasis on its potential role in breast cancer. Together, these studies will enhance our understanding of the molecular mechanisms of signal transduction by a potentially novel tumor suppressor gene and its roles in vivo. They will provide potentially novel therapeutic targets for diseases such as cancer.